This invention relates to methods for extracting fractions from material of biological origin. The fractions potentially contain one or more compounds which exhibit biological activity. The methods feature supercritical and near critical fluids.
At the present time, a number of major pharmaceutical companies are actively carrying out programs for the discovery of drugs from natural products. Typically, such programs involve screening of a large number of natural materials for therapeutic or other biological activity. These biomass materials may be obtained or derived from plant, animal, or microbial sources. Screening is carried out by assaying samples for indications of, for example, cytotoxicity, antibacterial activity, or antiviral activity.
In preparation for screening, the biomass is typically exposed to an extraction step. In many cases, however, the bioactive materials of interest may be sequestered within the substrate and not accessible to extraction. Thus, depending upon the particular biomass, the extraction may be facilitated by a preliminary size reduction (comminution) or disruption step. Comminution/disruption methods include for example grinding, sonication, and homogenization. Conventional comminution/disruption methods may have the disadvantages of incomplete disruption and/or product deterioration. They may also be time-consuming and expensive.
For the extraction, the biomass is contacted with a solvent such as butanol or ethyl acetate, so that compounds of potential interest may migrate from the biomass substrate to the solvent phase. Sometimes multiple extraction steps may be carried out on a single batch of biomass. A xe2x80x9cfractionxe2x80x9d refers to the material recovered from the biomass in a single one of these extraction steps. Fractions from the extraction steps are further processed so that they may be assayed for the activity of interest. Because different extraction methods will produce different extract profiles, there is a continuing interest in the development of new extraction techniques.
Conventional solvents are not always ideal for biomass extractions. These solvents can be difficult to remove from the compounds potentially exhibiting bioactivity, and also may extract a mixture of compounds which can mask bioactivity in an assay. The solvents may not penetrate membranes or other cellular structures surviving disruption. The solvation properties of conventional solvents cannot be readily modified by changing temperature, pressure, or the concentration of modifying cosolvents, and thus may be cumbersome to use when it is desired to carry out certain types of fractionations such as the selective extraction of compounds of varying polarity.
It would be highly desirable to have a method for fractional extraction of biomass constituents, including those which are sequestered within cells. It would be highly desirable to have a solvent system which can be readily modified by physical parameters and the addition of modifying cosolvents to selectively extract compounds of varying polarity, volatility, or hydrophilicity. It would be highly desirable to have a physical-chemical disruption process that provides for a high level of disruption without loss of active material. It would be desirable to disrupt biomass and form one or more fractions using a single apparatus.
Aspects of the present invention employ materials known as supercritical fluids. A material becomes a supercritical fluid at conditions which equal or exceed both its critical temperature and critical pressure. These parameters are intrinsic thermodynamic properties of all sufficiently stable pure compounds and mixtures. Carbon dioxide, for example, becomes a supercritical fluid at conditions which equal or exceed its critical temperature of 31.1xc2x0 C. and its critical pressure of 72.8 atm (1,070 psig). In the supercritical fluid region, normally gaseous substances such as carbon dioxide become dense phase fluids which have been observed to exhibit greatly enhanced solvating power. At a pressure of 3,000 psig (204 atm) and a temperature of 40xc2x0 C., carbon dioxide has a density of approximately 0.8 g/cc and behaves much like a nonpolar organic solvent, having a dipole moment of zero debyes. A supercritical fluid uniquely displays a wide spectrum of solvation power as its density is strongly dependent upon temperature and pressure. Temperature changes of tens of degrees or pressure changes by tens of atmospheres can change a compound""s solubility in a supercritical fluid by an order of magnitude or more. This unique feature allows for the fine-tuning of solvation power and the fractionation of mixed solutes. The selectivity of nonpolar supercritical fluid solvents can also be enhanced by addition of compounds known as modifiers (also referred to as entrainers or cosolvents). These modifiers are typically somewhat polar organic solvents such as acetone, ethanol, methanol, methylene chloride or ethyl acetate. Varying the proportion of modifier allows a wide latitude in the variation of solvent power.
In addition to their unique solubilization characteristics, supercritical fluids possess other physicochemical properties which add to their attractiveness as solvents. They can exhibit liquid-like density yet still retain gas-like properties of high diffusivity and low viscosity. The latter increases mass transfer rates, significantly reducing processing times. Additionally, the ultra-low surface tension of supercritical fluids allows facile penetration into microporous materials, increasing extraction efficiency and overall yields.
While similar in many ways to conventional nonpolar solvents such as hexane, it is well-known that critical fluid solvents can extract a different spectrum of materials than conventional techniques. Product volatilization and oxidation as well as processing time and organic solvent usage can be significantly reduced with the use of supercritical fluid solvents.
A material at conditions that border its supercritical state will have properties that are similar to those of the substance in the supercritical state. These so-called xe2x80x9cnear criticalxe2x80x9d fluids are also useful for the practice of this invention. For the purposes of this invention, a near critical fluid is defined as a fluid which is (a) at a temperature between its critical temperature (Tc) and 75% of its critical temperature and at a pressure at least 75% of its critical pressure, or (b) at a pressure between its critical pressure (Pc) and 75% of its critical pressure and at a temperature at least 75% of its critical temperature. In this definition, pressure and temperature are defined on absolute scales, e.g., Kelvins and psia. Table 1 shows how these requirements relate to some of the fluids relevant to this invention. To simplify the terminology, materials which are utilized under conditions which are supercritical, near critical, or exactly at their critical point will jointly be referred to as xe2x80x9ccriticalxe2x80x9d fluids.
The present invention utilizes critical fluids to fractionate biomass materials in two steps. In the first step, the biomass is disrupted by exposure to the critical fluid. It is hypothesized that this disruption involves at least two mechanisms, the first being liberation of structural constituents to cause permeability. The structural constituents are not necessarily solvated in the critical fluid, i.e., they may remain in the phase containing the biomass, but in any case lose their structural function in the biomass. For example, cell envelope constituents may be removed or liberated from a cell envelope, or waxy materials may be removed or liberated from plant biomass. The resulting permeability of the biomass makes certain contents of the biomass accessible to be extracted in subsequent steps. The second mechanism of disruption involves an explosive phenomenon due to the expanding critical fluid upon depressurization of the biomass. In the latter case, rapid decompression is sometimes desirable. Larger systems may require longer to decompress than smaller systems. In the former case, decompression is not required to provide the desired disruption. The nature of the biomass determines the relative importance of the two disruption mechanisms in any given application. During this first disruption step, an extract fraction may optionally be collected from the critical fluid contacting the biomass. In the second step of the fractionation, the disrupted biomass is subjected to a multiplicity of critical fluid extraction steps, the steps being characterized in that different solvation conditions are used in each. Thus, fractionation of the biomass is effected. As mentioned, critical fluid solvation properties may be varied by adjusting pressure, temperature, or modifier concentration. These parameters may be adjusted individually or in combination. Solvation conditions may also be varied through the use of different modifiers in a single fractionation procedure, although this would not typically be advantageous.
Preferably, each subsequent critical fluid is altered to change the solvation properties of the extracting fluid, so that each step can recover a different spectrum of compounds. The solvation properties of critical fluids can be altered by changing the temperature or pressure of the fluid. By way of example, a preferred temperature and pressure for a critical fluid comprising carbon dioxide is a temperature in the range of 10 to 60xc2x0 C. and a pressure in the range of 2000 to 4000 psig.
Preferred critical fluids comprise carbon dioxide, nitrous oxide, ethylene, ethane, propane and freons. The fluid may also contain modifiers. Preferred modifiers are methanol, ethanol, propanol, butanol, methylene chloride, ethyl acetate and acetone.
A preferred modifier comprises methanol. In one preferred embodiment, each subsequent extraction employs a larger concentration of methanol. Thus, the plurality of critical fluids becomes increasingly more hydrophilic. The first extraction step tends to remove lipophilic compounds while the last extraction step tends to remove hydrophilic compounds. Removal of the lipophilic materials allows the next more hydrophilic critical fluid to have access to more hydrophilic compounds trapped in cellular structures. Preferred methanol concentration ranges for a first extraction step on disrupted biomass, based on carbon dioxide at a pressure of 3000 psig and a temperature of 40xc2x0 C., are 0-5 volume %. For the same temperature and pressure, 5-10 volume % methanol is preferred for a second extraction step; 10-20 volume % methanol is preferred for a third extraction step; 20-30 volume % methanol is preferred for a fourth extraction step; 30-50 volume % methanol is preferred for a fifth extraction step.
Surprisingly and unexpectedly, the combination of disruption and extraction with critical fluids produces larger numbers of fractions exhibiting biological activity than corresponding fractions derived from conventional organic solvent extractions. The use of critical fluids allows for easy removal of much of the solvent by mere depressurization. Use of a single apparatus to perform both the disruption and extraction steps minimizes labor and increases efficiency. Indeed, the entire process can be readily automated. The use of critical fluids allows the extraction conditions to be readily varied by temperature, pressure, or modifier solvents. Use of critical fluids for both the disruption and extraction simplifies the procedure and minimizes equipment needs, processing time, potential for contamination, and loss of yield. These and other features and advantages will be readily apparent from the drawing and detailed discussion which follow.